This invention relates to the gene and CDNA of a as porcine derived CNP (C-type natriuretic peptide), as well a porcine derived CNP precursor protein.
Peptides assignable to two different peptide families that are named "atrial natriuretic peptide (ANP)" and "brain natriuretic peptide (BNP)" have recently been discovered as hormones or nerve transmitters that regulate the homeostatic balance of body fluid volume and blood pressure in vivo. The structures of those peptides, the mechanism of their biosynthesis, as well as their physiological actions have also been unravelled.
Very recently, the present inventors discovered from porcine brain a novel peptide that was named "C-type natriuretic peptide (CNP)" and that belonged to a third family of peptides.
The first clue to the discovery of ANP was reported by de Bold et al. in 1981. Finding that significant diuresis occurred when a rat's atrial crude extract was injected intravenously into another rat, de Bold et al. reported the existence of a natriuresis promoting factor in the atrium (de Bold, A. J. et al., Life Sci., 28, 89, 1981). Kangawa et al. later isolated that factor from human atrium, unravelled its structure and named it "atrial natriuretic peptide (ANP)" (Kangawa, K. et al., Biochem. Biophys. Res. Commun., 118, 131, 1984; Kangawa, K. et al., Nature, 313, 397, 1985; Japanese Patent Publication No. 19520/1988; Japanese Patent Public Disclosure No. 184098/1985 and 260596/1985). It has been found that human ANP (hANP) as it occurs in the atrium is classified into three types, .alpha.-, .beta.- and .gamma.-types, according to the molecular weight; .alpha.-type hANP (.alpha.-hANP) is a single-stranded peptide that consists of 28 amino acids having a single S--S bond in the molecule; .beta.-type hANP (.beta.-hANP) is an antiparallel dimer having an S--S bond formed between the molecules of .alpha.-hANP; and .gamma.-type hANP (.gamma.-hANP) is a high-molecular weight protein composed of 126 amino acids, with x-hANP being contained in the C-terminal portion. Further, CDNA for hANP has been isolated and the routes of biosynthesis of .alpha.-, .beta.- and .gamma.-hANP have been identified the basis of analysis of that CDNA, leading to the conclusion that each of those three types of hANP is biosynthesized from a common precursor protein (Oikawa, S. et al., Nature, 309, 724, 1984).
It is already known that among the three types of hANP, .alpha.-hANP is chiefly secreted into blood.
Ever since the structure of hANP was first unravelled, the structures of ANPs derived from other mammals have also been studied (Japanese Patent Public Disclosure Nos. 184097/1985 and 7298/1986) and today the following knowledge is available: ANPs have similar amino acid sequences over a broad spectrum of mammals ranging from rodents to humans; .alpha.-type ANP (.alpha.-ANP) has the same amino acid sequence in higher mammals including humans, dogs and pigs; and .alpha.-type ANPs derived from rats and rabbits have entirely the same amino acid sequence as .alpha.-hANP except that the methionine residue in position 12 is replaced by an isoleucine residue (Oikawa, S. et al., Biochem. Biophys. Res. Commun., 132, 892, 1985; Forssmann, W. G. et al., Anat. Embryol., 168, 307, 1983).
The first ANP isolate was obtained from the atrium but later studies involving the preparation of anti-ANP antibodies and examination of their distribution in vivo have shown that ANP also occurs in the brain as well as in the atrium, except that in the brain the N-terminus of .alpha.-ANP is cut off to yield shorter .alpha.-ANP [4-28] and .alpha.-ANP [5-28] (Ueda, S. et al., Biochem. Biophys. Res. Commun., 149, 1055, 1987). Since ANP-containing neurons have been reported to occur in the hypothalamus and pontine tegmentum of the brain (Cantin, M. et al., Histochemistry, 80, 113, 1984; Saper, C. B. et al., Science, 227, 1047, 1985), it is speculated today that ANP may also work in the brain as a nerve transmitter that participates in the regulation of the cardiovascular system. The physiological actions of ANP are diverse and are not limited to a marked natriuretic action alone; it has recently been found that it is capable of not only lowering the blood pressure but also suppressing the production of aldosterone from the adrenal cortex. It is therefore clear today that ANP as it is secreted from the atrium into blood not only works as a hormone that regulates the homeostatic balance of body fluid volume and blood pressure but that in the brain it also works as a nerve transmitter for the nerve system to regulate the homeostatic balance of body fluid volume and blood pressure.
Brain natriuretic peptide (BNP) was first isolated from porcine brain and identified by Sudoh et al., in 1988 (Sudoh, T. et al., Nature, 332, 78, 1988). The first BNP isolate (pBNP-26) is a peptide that consists of 26 amino acid residues having a single S--S bond in the molecule and although it is similar to ANP in structure, i.e., in terms of primary amino acid sequence and the mode of S--S binding (producing a ring structure composed of 17 amino acid residues), BNP is clearly distinguishable from ANP. As in the case of ANP, natriuretic and hypotensive actions have been verified for BNP, which has therefore been named "brain natriuretic peptide (BNP)". At a later time, pBNP-32 composed of 32 amino acid residues having 6 amino acids attached to the N-terminus-of pBNP-26 was isolated from porcine brain (Sudoh, T. et al., Biochem. Biophys. Res. Commun., 155, 726, 1988); from porcine atrium, a peptide named ".gamma.Y-BNP" which was composed of 106 amino acids was also isolated and identified (Minamino, N. et al., Biochem. Biophys. Res. Commun., 157, 402, 1988).
As of today, the cDNAs of human and rat BNPs have been isolated and the structures of precursors for those BNPs have also become clear (Sudoh, T. et al., Biochem. Biophys. Res. Commun., 159, 1427, 1989; Kojima, M. et al., Biochem. Biophys. Res. Commun., 159, 1420, 1989).
On the basis of these results, it has been found that the peptides of the BNP family are biosynthesized from precursors that are entirely different from ANP.
As already mentioned, BNP was first isolated from the brain. It was later found that BNP was present in porcine brain in an amount ten times as much as ANP and that like ANP, BNP also occurred in the atrium (though in an amount of only 2-3% of ANP) to be secreted into blood (Minamino, N. et al., Biochem. Biophys. Res. Commun., 155, 740, 1988; Aburaya, M. et al., Biochem. Biophys. Res. Commun., 165, 872, 1989). From these facts, it was found that like ANP, BNP worked as a nerve transmitter in the brain and also worked as a hormone to be secreted from the atrium into blood, in either case helping regulate the homeostatic balance of body fluid volume and blood pressure. As exemplified by natriuretic peptides, not a single peptide but a plurality of peptides can participate in the regulation of a certain physiological action in vivo (e.g. homeostasis of body fluid volume and blood pressure) and opioid peptide, tachykinin and endothelin have so far been recognized as other examples of such peptides. It has been known that three different families exist for each of those peptides (Hollt, V., Trend Neuro Sci., 6, 24, 1983; Nakanishi, S., Physiol. Review, 67, 1117, 1987; Inoue, A. et al., Proc. Natl. Acad. Sci., U.S.A., 86, 2863, 1989). This had increased the possibility that aside from the natriuretic peptides so far been known to be assignable to ANP and BNP families, peptides that could be classified into a third family might exist. In this regard, the present inventors very recently succeeded in discovering two novel peptides from porcine brain that did not belong to either ANP or BNP family but that belonged to a third family of natriuretic peptides. Those peptides were named "C-type natriuretic peptide (CNP)". The first discovered CNP was a peptide composed of 22 amino acid residues (this peptide is hereinafter abbreviated as "CNP-22"); the structure of this peptide is similar to but clearly distinguishable from those of ANP and BNP. Stated more specifically, CNP-22 is similar to ANP and BNP in that it has a ring structure composed of 17 amino acid residues based on intramolecular S--S binding and that the primary amino acid sequence forming this ring structure in CNP-22 is highly homologous to that in .alpha.-ANP and BNP-32. As a matter of fact, 12 out of the 17 amino acid residues was identical among those three peptides. However, except for the ring structural portion, CNP-22 is entirely different from .alpha.-ANP and BNP-32 in N- and C-terminal portions. A particularly characteristic feature is found in the structure of the C-terminal portion; in the case of .alpha.-ANP, 5 amino acid residues (in the case of BNP-32, 6 amino acid residues) are present at the C-terminus of the cysteine residue forming the ring structure, thereby producing a "tail" structure, but no such "tail" structure is present in CNP-22 since its C-terminus is a cysteine residue.
As described above, CNP-22 has an obviously different structure than .alpha.-ANP and BNP-32; in addition, it has been verified that when administered to rats, CNP-22 exhibits obvious natriuretic and hypotensive actions it has therefore been found that CNP-22 is a new peptide assignable to a third family of natriuretic peptides in vivo (Japanese Patent Application No. 105047/1990). The present inventors later prepared anti-CNP-22 antibodies and purified from porcine brain those peptides which exhibited immunoreactivity with those antibodies. As a result, the present inventors successfully isolated a peptide named "CNP-53". An analysis of its structure showed that CNP-53 was a peptide composed of 53 amino acid residues containing CNP-22 in the C-terminus, namely, a peptide having 31 additional amino acid residues attached to the N-terminus of CNP-22 (see the commonly assigned patent application filed on the same date as the subject application).
In short, the following observations have been obtained to date: at least three families (ANP family, BNP family and CNP family) of natriuretic peptides having obviously different structures occur in mammals; peptides of the ANP and BNP families are not only secreted from the atrium into blood and work as hormones that regulate the homeostatic balance of body fluid volume and blood pressure; they are also biosynthesized in the brain, where they work as nerve transmitters for the nerve system to regulate the homeostatic balance of body fluid volume and blood pressure. However, the recently discovered peptides of the CNP family (CNP-22 and CNP-53) occur in so smaller amounts in the brain than ANP and BNP that as of today no detailed information has been obtained with respect to the mechanism of CNP biosynthesis, their distribution in vivo and physiological actions.